Joint bioscaffolds

ABSTRACT

Provided herein are compositions and medical devices, and in particular, biodegradable scaffolds capable of repairing and replacing cartilagenous meniscuses. Also provided herein are methods of using scaffolds for treating degenerative tissue disorders. In certain embodiments, such scaffolds can promote tissue regeneration of a temporal mandibular joint (TMJ) meniscus.

This application claims the benefit under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 61/156,162, filed Feb. 27, 2009,which is incorporated herein by reference in its entirety.

This invention was made with government support under Grant #SAP4100045998 awarded by the Commonwealth of Pennsylvania. The governmenthas certain rights in the invention.

The temporomandibular joints (TMJs) connect the jaw to the skull andgenerate large amounts of force in the jaw. In between these two bonesrests a fibrocartilagenous disc termed the TMJ meniscus, which acts todisperse the forces on the jaw and reduce friction during movement. Inanatomy, a meniscus is a fibrocartilagenous structure present, e.g., inthe knee, acromioclavicular, and sternoclavicular joints that, incontrast to articular discs, only partly divides a joint cavity.

The TMJs are unusual because they are one of the only synovial joints inthe human body comprising a disc meniscus. The disc separates the lowerjoint compartment formed by the mandible and the articular disc(allowing rotational movement) from the upper joint compartment(allowing translational movements). The top of the mandible which matesto the under-surface of the disc is termed the “condyle” and thetemporal bone of the skull that mates to the upper surface of the diskis termed the “glenoid (or mandibular) fossa.” The TMJ meniscus differssubstantially from other meniscuses such as the knee meniscus, as itcomprises almost entirely type I collagen as opposed to approximately80% type 1120% type II collagen in the knee.

Temporomandibular joint disorder or dysfunction (TMD) occurs when thereis pain at or near the temporomandibular joint. Broadly, TMD comprises agroup of disorders involving the joints, muscles, tendons, ligaments,and blood vessels at the joint. One type of disorder of the TMJ isinternal derangement (ID), which involves an abnormality of themeniscus-temporal fossa relationship, resulting in a mechanical disorderthat creates irregular joint noises and prohibits normal condylarmovement. Although the etiology remains obscure, various inflammatorymediators have been implicated. For example, synovial fluid analysisindicates a role of cytokines and proteinases in development of ID.Moreover, interleukins have been detected in both ID and rheumatoidarthritis of the TMJ. One possible mechanism is the inducing release ofproteinases and collagenases by inflammatory cytokines. Despite thisunderstanding, treatment for such derangement is usually surgical.

For example, internal derangement due to ankylosis, meniscalperforation, and degenerative joint disease, among others, can betreated by a meniscectomy Complications arising from meniscectomywithout replacement include heterotopic bone formation and jointankylosis. The rationale for replacing the TMJ meniscus with asubstitute material is to protect the articular surfaces from furtherdegenerative changes and to avoid joint adhesion formation. Manyalloplastic materials such as SILASTIC™, silicone and PROPLAST™-Teflon,have been used to replace the TMJ meniscus but results have been lessthan satisfactory. Often times, joint pathology is more severe followingthe placement of such devices. Autograft tissues have been used both asdisc replacement materials following meniscectomy and as interpositionalmaterials in the treatment of joint ankylosis. Sources such as thetemporalis muscle flap, auricular cartilage, and dermis have proven farbetter options than their alloplastic counterparts but still have theobvious disadvantage of morbidity associated with the graft donor site.Furthermore, a variety of studies have shown fibrosis or, in the case ofthe temporalis muscle flap, necrosis and devitalization of autogenoustissue grafts.

Thus, what is needed is a graft material for the treatment of TMJpathology with associated meniscus abnormality is a scaffold forcellular influx and that would be readily implanted without theassociated morbidity of autogenous tissue harvest. It is also desirablethat the graft closely match the natural state of the disc which ishypovascular, aneural, and alymphatic while being able to functionmechanically immediately after implantation.

SUMMARY

Extracellular matrix (ECM) scaffolds for cartilagenous tissueregeneration and replacement are disclosed herein. In certainembodiments the scaffolds comprise a particulate and/or gel ECM coreencapsulated within an ECM sheath. The core can be aparticulate/powdered material, a gel material or both, and in each case,the core and/or the sheath optionally comprise cells, such as one ormore of stem cells, progenitor cells and differentiated cells, such asfibroblasts and chondrocytes. The encapsulation of particulate ECMallows for the structure to operate in three-dimensions as well asdeform elastically in response to compressive and shear forces, and,thus, respond to biomechanical stresses much like the original bodilytissues. The compositions are compatible with any desired shapeconsistent with any suitable method.

In certain embodiments, the scaffold can be tailored to the particularanatomy of an individual and thus can be designed to operate as animplant device. As such, it can operate as an implant device whichreplaces fibrocartilagenous meniscuses in the temporomandibular joints(TMJs) and also provides a substrate onto which cartilage producingcells can attach and repair and/or replace the original joint meniscus.One particularly useful replacement is of a temporomandibular jointmeniscus following meniscectomy.

In yet other embodiments, ECM scaffolds comprise particulate and/or gelECM encapsulated within a sheath comprising xenogenic or allogeneic ECMsheets. The particulate ECM and ECM sheaths comprise both the structuraland functional proteins present within native mammalian ECM. The ECM maybe derived from mammalian tissue sources such as, without limitation,the urinary bladder, esophagus, skin, liver, spleen, heart, pancreas,ovary, and blood vessels. Sources of the ECM may include, withoutlimitation, any warm blooded vertebrate, such as pig, cow, horse, ormonkey. In one embodiment, both the particulate ECM and theencapsulating ECM sheath are derived from porcine urinary bladder matrix(UBM).

As disclosed herein, ECM compositions facilitate influx of cells intothe implanted devices, thereby constructively remodeling the ECM deviceinto fibrocartilagenous tissue that conforms to the desired shape.Likewise a method of promoting wound healing or tissue generation orregeneration in a patient is provided comprising contacting an implantas described herein with cells in vitro (for instance, ex vivo forautologous cells), culturing the cells in vitro so that the cells growin and/or on the scaffold. Useful cell types include fibroblasts,chondrocytes and stem or progenitor cells.

In addition, surgical methods of replacing a joint meniscus and treatingtemporomandibular joint disease are provided. In a one embodiment, atemporo-mandibular joint meniscus is removed and replaced with athree-dimensional tissue scaffold comprised of a particulate and/or gelECM core encapsulated within an ECM sheath. Thus, the scaffold acts as astructurally and functionally normal meniscus while facilitating influxof cells into the scaffold. In the one embodiment, the temporomandibularjoint meniscus is replaced or repaired.

According to certain embodiments, the scaffolds are biodegradable,elastomeric, porous and biocompatible. Also provided are methods ofpreparing biodegradable elastomeric scaffolds and promoting woundhealing and/or tissue regeneration within a patient. The methodcomprises implanting a biodegradable elastomeric scaffold at, around ornear a site in need of wound healing, tissue remodeling and/or tissueregeneration. In another non-limiting embodiment, such a scaffoldcomprises cells. For example and without limitation, such a methodcomprises culturing cells in and/or on a biodegradable elastomericscaffold in vitro and implanting the scaffold. In yet anothernon-limiting embodiment, the biodegradable elastomeric scaffoldcomprises bioactive or therapeutic agents, such as, without limitationgrowth factors, antibiotics, and anti-inflammatory agents.

According to certain non-limiting embodiments, the biological polymericcomponent is an extracellular matrix material. The ECM-derived materialmay be isolated from, for example and without limitation, urinarybladder tissue. In one non-limiting embodiment, the extracellular matrixmaterial comprises decellularized epithelial basement membrane andsubjacent tunica propria. In another embodiment, the extracellularmatrix-derived material comprises tunica submucosa. In yet anotherembodiment, extracellular matrix-derived material comprises epithelialbasement membrane, subjacent tunica propria and tunica submucosa. Incertain non-limiting embodiments, the extracellular matrix-derivedmaterial is isolated from small intestinal submucosa or the dermis ofskin.

In certain embodiments, the device resembles a hat in which there is acore center section and a brim which extends from the core. In suchstructures, three ultrastructural parameters operate in unison to allowan articulating device to function even from the moment of implantation:the height and composition of the core which modulates compressiveforce, the diameter of circular core, including the major and minordiameters where the core is elliptical, which modulates the lateraldisplacement at the site of implantation, and the radius of the brimwhich modulates the type and number of attachment points for the devicein the implant site. Moreover, the functionality of the device is notdegraded over time, as the microstructure allows for influx of cells,thus allowing the body to remodel the device to the unique anatomy ofeach implanted individual.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the normal articulation of the TMJ meniscus (M) inrelation to the condyle (C) and the mandibular fossa (MF) during jawmovement.

FIG. 1B shows the deranged articulation of the TMJ meniscus where ananterior dislocation of the meniscus with reduction has occurred.

FIG. 1C shows the deranged articulation of the TMJ meniscus where ananterior dislocation of the meniscus without reduction has occurred.

FIG. 2 shows schematically a cross-sectional view of the wall of theurinary bladder (not drawn to scale). The following structures areshown: epithelial cell layer (A), basement membrane (B), tunica propria(C), muscularis mucosa (D), tunica mucosa (E), tunica muscularis externa(F), tunica serosa (G), tunica mucosa (H), and lumen of the bladder (L).

FIG. 3 shows a schematic of one embodiment of a TMJ meniscus replacementdevice comprising a central core 110 a brim 120 and suture attachmentpoints 130 (holes) in the brim. The device may be attached next to theskull laterally and medially.

FIG. 4 shows a top-down schematic of one embodiment of a TMJ meniscusreplacement device comprising a central core 110 and a brim 120 having amajor axis 5 and a minor axis 6.

FIG. 5 shows a side-view schematic of one embodiment of a TMJ meniscusreplacement device comprising an elliptical central core 200 a brim 220encased in a sheath 230 and 240 covering the particulate or gel 215 core210 and extending into the brim 220.

FIG. 6 shows a side-view schematic of one embodiment of a TMJ meniscusreplacement device comprising a circular central core 200 a brim 220encased in a sheath 230 and 240 covering the particulate or gel 215 core210 and extending into the brim 220.

FIG. 7 is a photograph of one embodiment of a TMJ meniscus replacementdevice prior to implantation.

FIG. 8 is a photograph of one embodiment of a TMJ meniscus replacementdevice after two months of implantation. Notably, cellular influx andvascularization of the core has occurred, indicating the body has begunto actively remodel the device.

FIG. 9 is a schematic for a non-limiting embodiment of a mold for acanine TMJ meniscus replacement device. All measurements are shown inmillimeters (mm). The molds creates multiple devices with multipledimensions at the same time. FIG. 9A shows a top-view of the mold. FIG.9B shows a side-view of the mold along the x-axis. FIG. 9C shows aside-view of the mold along the y-axis.

FIG. 10 is a schematic for a non-limiting embodiment of a mold for ahuman TMJ meniscus replacement device. All measurements are shown inmillimeters (mm). FIG. 10A shows a top-view of the mold. FIG. 10B showsa side-view of the mold along the x-axis.

FIG. 10C shows a side-view of the mold along the y-axis.

FIG. 11 is a photograph of the UBM meniscus in dog 1 described inExample 4 after six months.

FIGS. 12A and 12B are photomicrographs of Hematoxylin and Eosin stainsfor the UBM meniscus for dogs 1 and 2 respectively.

FIG. 13A is a photograph of a cross-section of the UBM meniscus from Dog1 showing gross morphology. FIGS. 13B and 13C are photomicrographs(200×) showing the results of Von Kossa staining of sections of the UBMmenisci from dogs 1 and 2.

FIGS. 14A and 14B are photomicrographs (200×) showing CD31 staining fordogs 1 and 2, respectively.

FIGS. 15A and 15B are photomicrographs (200×) showing CD68 staining fordogs 1 and 2, respectively.

FIGS. 16A and 16B are graphs showing compressive behavior for a nativecanine TMJ disc and for a pre-implantation UBM construct, respectively.

FIGS. 17A and 17B are graphs showing the relaxation data for nativecanine TMJ and preimplanted UBM, respectively.

FIG. 18 is a graph showing percent relaxation for native canine TMJ,pre-implantation UBM and remodeled UBM scaffold at 6 monthspost-implantation.

FIG. 19 is a graph showing the Tangent Modulus for native canine TMJ,pre-implantation UBM and remodeled UBM scaffold at 6 monthspost-implantation.

FIG. 20 is a graph showing Collagen and GAG content for native canineTMJ, pre-implantation UBM and remodeled UBM scaffold at 6 monthspost-implantation.

FIG. 21 is a graph showing water content for native canine TMJ,pre-implantation UBM and remodeled UBM scaffold at 6 monthspost-implantation.

FIG. 22A is a tabulation of the data from Example 2. FIG. 22B providesaverages and standard deviation.

DETAILED DESCRIPTION

Described herein are scaffolds suitable for use in tissue engineeringand regenerative medicine applications, such as replacement offibrocartilagenous tissue. Such scaffolds are useful for replacing andrepairing cartilagenous discs such as the temporomandibular jointmeniscus. Generally, any material that is biocompatible, biodegradable,and has mechanical properties similar to that of native tissue can beused as a scaffold, including for example elastomeric scaffolds. In oneembodiment, the scaffold comprises a powdered biological extracellularmatrix (ECM) encased in a laminar sheath of ECM. In yet anotherembodiment, the device consisting of particulate ECM derived fromporcine urinary bladder (UBM-ECM) is encased within sheets of UBM-ECM tomimic the shape and size of the native TMJ meniscus. In anothernon-limiting embodiment, the scaffold comprises bioactive or therapeuticagents.

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges are both preceded by the word “about”. In this manner,slight variations above and below the stated ranges can be used toachieve substantially the same results as values within the ranges.Also, unless indicated otherwise, the disclosure of these ranges isintended as a continuous range including every value between the minimumand maximum values. For definitions provided herein, those definitionsrefer to word forms, cognates and grammatical variants of those words orphrases.

Scaffolds can be used for a large number of medical applicationsincluding, but not limited to, wound healing, tissue remodeling, andtissue regeneration. For example and without limitation, the scaffoldcan be used for wound healing. In one non-limiting embodiment, thescaffold comprises bioactive agents to facilitate tissue healing, tissueremodeling and/or angiogenesis. In another non-limiting embodiment, thescaffold comprises bioactive agents to ward off bacteria and otherpathogens, recruit selected cell types, such as stem cells, or inducedifferentiation of cells. In yet another non-limiting embodiment, thescaffold comprises pores to allow a wound to drain or for cells to passthrough and deposit connective tissue. In yet another non-limitingembodiment, the scaffold comprises combinations of cells and bioactiveagents. In another non-limiting embodiment, combinations of cells andbioactive agents are added to the scaffold before or during implantationat a site in a patient.

As used herein, the term “polymer” refers to both synthetic polymericcomponents and biological polymeric components. The scaffolds describedherein can comprise any suitable combination of synthetic polymericcomponents and biological polymeric components. “Biological polymer(s)”are polymers that can be obtained from biological sources, such as,without limitation, mammalian or vertebrate tissue, as in the case ofcertain extracellular matrix-derived (ECM-derived) compositions.Biological polymers can be modified by additional processing steps.Polymer(s), in general include, for example and without limitation,mono-polymer(s), copolymer(s), polymeric blend(s), block polymer(s),block copolymer(s), cross-linked polymer(s), non-cross-linkedpolymer(s), linear-, branched-, comb-, star-, and/or dendrite-shapedpolymer(s), where polymer(s) can be formed into any useful form, forexample and without limitation, a hydrogel, a porous mesh, a fiber,woven mesh, or non-woven mesh, such as, for example and withoutlimitation, a non-woven mesh formed by electrodeposition.

Generally, the polymeric components suitable for the scaffold describedherein may be any polymer that is biodegradable and biocompatible. By“biodegradable”, it is meant that a polymer, once implanted and placedin contact with bodily fluids and/or tissues, will degrade eitherpartially or completely through chemical, biochemical and/or enzymaticprocesses. Non-limiting examples of such chemical reactions includeacid/base reactions, hydrolysis reactions, and enzymatic cleavage. Incertain non-limiting embodiments, the biodegradable polymers maycomprise homopolymers, copolymers, and/or polymeric blends comprising,without limitation, one or more of the following monomers: glycolide,lactide, caprolactone, dioxanone, and trimethylene carbonate.Non-limiting examples of biodegradeable polymers include poly(esterurethane) urea elastomers (PEUU) and poly(ether ester urethane) ureaelastomers (PEEUU). In other non-limiting embodiments, the polymer(s)comprise labile chemical moieties, non-limiting examples of whichinclude esters, anhydrides, polyanhydrides, or amides, which can beuseful in, for example and without limitation, controlling thedegradation rate of the scaffold and/or the release rate of therapeuticagents from the scaffold. Alternatively, the polymer(s) may containpeptides or biomacromolecules as building blocks which are susceptibleto chemical reactions once placed in situ. In one non-limiting example,the polymer is a polypeptide comprising the amino acid sequencealanine-alanine-lysine, which confers enzymatic lability to the polymer.In another non-limiting embodiment, the polymer composition may comprisea biomacromolecular component derived from an ECM. For example, thepolymer composition may comprise the biomacromolecule collagen so thatcollagenase, which is present in situ, can degrade the collagen.

The polymer components may be selected so that they degrade in situ on atimescale that is similar to an expected rate of healing of the wound ortissue. Non-limiting examples of in situ degradation rates includebetween one week and one year or increments therebetween for instance,between two weeks and 10 months, and between one month and six month.

The polymeric components used to make the devices disclosed herein arepreferably biocompatible. By “biocompatible,” it is meant that a polymercomposition and its normal in vivo degradation products arecytocompatible and are substantially non-toxic and non-carcinogenic in apatient within useful, practical and/or acceptable tolerances. By“cytocompatible,” it is meant that the polymer can sustain a populationof cells and/or the polymer composition, device, and degradationproducts, thereof are not cytotoxic and/or carcinogenic within useful,practical and/or acceptable tolerances. For example, the polymer whenplaced in a human epithelial cell culture does not adversely affect theviability, growth, adhesion, and number of cells. In one non-limitingembodiment, the compositions, and/or devices are “biocompatible” to theextent they are acceptable for use in a human patient according toapplicable regulatory standards in a given jurisdiction. In anotherexample the biocompatible polymer, when implanted in a patient, does notcause a substantial adverse reaction or substantial harm to cells andtissues in the body, for instance, the polymer composition or devicedoes not cause necrosis or an infection resulting in harm to tissuesfrom the implanted scaffold.

The mechanical properties of a biodegradable scaffold can be optimizedto operate under the normal strain and stress on the native tissue atthe site of implantation. In certain non-limiting embodiments, themechanical properties of the scaffold are optimized similar to oridentical to that of native soft tissue, such as fascia, connectivetissue, blood vessel, muscle, tendon, fat, etc.

The mechanical properties of the scaffold also may be optimized to besuitable for surgical handling. In one non-limiting embodiment, thescaffold is flexible and can be sutured to the site. In another, thescaffold is foldable and can be delivered to the site by minimallyinvasive laparoscopic methods.

In one non-limiting example, biodegradable scaffolds can be surgicallyimplanted to replace a TMJ meniscus. The implant can be placed by makinga preauricular incision while the patient is under general anesthesia.Prior to surgery, the anterior-posterior (“AP”) dimension of thecondylar head and glenoid fossa can be estimated and then duringsurgery, the size can be measured for the implant. The implant can beinserted over the condylar head and fixed with slow-resorbing sutures(for example, either 4.0 MERSILINE™ or 4.0 CAPROSYN™, among others) tothe zygomatic bone (e.g., the inferolateral surface of the articulatingfossa). In certain embodiments, the implant can be secured with threefixation sutures to the zygomatic arch. For example, three holes can becreated through the inferolateral surface of the zygomatic bonecoincident with the anterior-posterior (AP) dimension of thearticulating fossa. Next, a narrow fissure-shaped bun in a rotaryinstrument under irrigation can be used to create fixation holes forpassage of the suture needle through the zygomatic bone. Once theimplantation site is prepared, the implant can be placed into thesuperior joint space and the suture needle can be passed through thelateral extension of the implant. Once in place, the medial brim of theimplant can be tucked into the medial aspect of the joint space. Forfixation, a suture needle can be placed through the holes in the boneand the brim of the implant, and then the suture can be used to securethe implant to the bone. Finally, the suture can be tied to secure theimplant in the fossa. In certain embodiments, one or two additionalsutures may be placed laterally through adjacent muscle tissue to addfurther stability of the implant.

The physical and/or mechanical properties of the biodegradable scaffoldcan be optimized according to the intended use. Variables that can beoptimized include without limitation, the extent of physical, chemicalor photooxidative cross-linking in a network comprising polymericcomponents, the ratio of polymeric components within the network, thedistribution of molecular weight of the polymeric components, and themethod of processing the polymers. Polymers are typicallysemicrystalline and their physical properties and/or morphology aredependant upon a large number of factors, including monomer composition,polydispersity, average molecular weight, cross-linking, andmelting/crystallization conditions. For example, flow and/or shearconditions during cooling of a polymer melt are known to affectformation of crystalline structures in the composition. In onenon-limiting embodiment, the scaffold comprises a polymeric componentthat provides strength and durability to the scaffold, yet iselastomeric so that the mechanical properties of the scaffold aresimilar to the native tissue surrounding the wound or site in need oftissue regeneration.

As described herein, according to certain non-limiting embodiments, oneor more of the polymeric components of the biodegradable scaffold iselastomeric. In one non-limiting example, the scaffold has physicalproperties similar to that of cartilage. In certain non-limitingembodiments, the biodegradable scaffold comprises highly distensiblepolymeric components. Examples of suitable polymers include those thathave a breaking strain ranging from about 100% to about 900%, includingany increments therebetween, for example between 200% and 800%, orbetween 325% and 600%. In other non-limiting embodiments, the breakingstrain of the polymer is between 50% and 100% including any incrementstherebetween. Further, it is often useful to select polymers withtensile strengths of from 10 kPa to 30 MPa, including incrementstherebetween, such as from 5 MPa to 25 MPa, and between 8 MPa and 20MPa. In certain non-limiting embodiments, the initial modulus is between10 kPa to 100 MPa and increments therebetween, such as between 10 MPaand 90 MPa, and between 20 MPa and 70 MPa.

The extracellular matrix is useful for promoting cell growth on thescaffold, recruiting appropriate host cells for construction,remodeling, and/or enhancement of biocompatibility. In one non-limitingembodiment, the biological polymeric component comprises and includes anextracellular matrix-derived material. As used herein, the terms“extracellular matrix” and “ECM” refer to a complex mixture ofstructural and functional biomolecules and/or biomacromoleculesincluding, but not limited to, structural proteins, specializedproteins, proteoglycans, glycosaminoglycans, and growth factors thatsurround and support cells within mammalian tissues and, unlessotherwise indicated, is acellular.

Generally, any type of extracellular matrix (ECM) can be used to preparethe biological, ECM-derived polymeric component of the biodegradableelastomeric scaffold (for example and without limitation, see U.S. Pat.Nos. 4,902,508; 4,956,178; 5,281,422; 5,352,463; 5,372,821; 5,554,389;5,573,784; 5,645,860; 5,771,969; 5,753,267; 5,762,966; 5,866,414;6,099,567; 6,485,723; 6,576,265; 6,579,538; 6,696,270; 6,783,776;6,793,939; 6,849,273; 6,852,339; 6,861,074; 6,887,495; 6,890,562;6,890,563; 6,890,564; and 6,893,666; each of which is incorporated byreference in its entirety). By “ECM-derived material” it is meant acomposition that is prepared from a natural ECM or from an in vitrosource wherein the ECM is produced by cultured cells and comprises oneor more polymeric components (constituents) of native ECM. ECMpreparations can be considered to be “decellularized” or “acellular”,meaning the cells have been removed from the source tissue throughprocesses described herein and known in the art.

According to one non-limiting example of the ECM-derived material, ECMis isolated from a vertebrate animal, for example, from a warm bloodedmammalian vertebrate animal including, but not limited to, human,monkey, pig, cow, sheep, etc. The ECM may be derived from any organ ortissue, including without limitation, urinary bladder, intestine, liver,heart, esophagus, spleen, stomach and dermis. The ECM can comprise anyportion or tissue obtained from an organ, including, for example andwithout limitation, submucosa, epithelial basement membrane, tunicapropria, etc. In one non-limiting embodiment, the ECM is isolated fromurinary bladder, which may or may not include the basement membrane. Inanother non-limiting embodiment, the ECM includes at least a portion ofthe basement membrane. In certain non-limiting embodiments, the materialthat serves as the biological component of the scaffold consistsprimarily (e.g., greater than 70%, 80%, or 90%) of ECM. In anothernon-limiting embodiment, the biodegradable elastomeric scaffold maycontain at least 50% ECM, at least 60% ECM, at least 70% ECM, and atleast 80% ECM. In yet another non-limiting embodiment, the biodegradableelastomeric scaffold comprises at least 10% ECM. The ECM material may ormay not retain some of the cellular elements that comprised the originaltissue such as capillary endothelial cells or fibrocytes. The type ofECM used in the scaffold can vary depending on the intended cell typesto be recruited during wound healing or tissue regeneration, the nativetissue architecture of the tissue organ to be replaced, the availabilityof the tissue source of ECM, or other factors that affect the quality ofthe final scaffold and the possibility of manufacturing the scaffold.For example and without limitation, the ECM may contain both a basementmembrane surface and a non-basement membrane surface, which would beuseful for promoting the reconstruction of tissue. In certainembodiments, an implantable device can comprise either a smooth basementmembrane surface (luminal) or a rough non-basement membrane surface(abluminal). For example, in applications where the device operateswithin a synovial joint, a smooth surface for the device can beparticularly advantageous.

In one non-limiting embodiment, the ECM is harvested from porcineurinary bladders (also known as urinary bladder matrix or UBM). Briefly,the ECM is prepared by removing the urinary bladder tissue from a pigand trimming residual external connective tissues, including adiposetissue. All residual urine is removed by repeated washes with tap water.The tissue is delaminated by first soaking the tissue in adeepithelializing solution, for example and without limitation,hypertonic saline (e.g. 1.0 N saline), for periods of time ranging fromten minutes to four hours. Exposure to hypertonic saline solutionremoves the epithelial cells from the underlying basement membrane.Optionally, a calcium chelating agent may be added to the salinesolution. The tissue remaining after the initial delamination procedureincludes the epithelial basement membrane and tissue layers abluminal tothe epithelial basement membrane. The relatively fragile epithelialbasement membrane is invariably damaged and removed by any mechanicalabrasion on the luminal surface. This tissue is next subjected tofurther treatment to remove most of the abluminal tissues but maintainthe epithelial basement membrane and the tunica propria. The outerserosal, adventitial, tunica muscularis mucosa, tunica submucosa andmost of the muscularis mucosa are removed from the remainingdeepithelialized tissue by mechanical abrasion or by a combination ofenzymatic treatment (e.g., using trypsin or collagenase) followed byhydration, and abrasion. Mechanical removal of these tissues isaccomplished by removal of mesenteric tissues with, for example andwithout limitation, Adson-Brown forceps and Metzenbaum scissors andwiping away the tunica muscularis and tunica submucosa using alongitudinal wiping motion with a scalpel handle or other rigid objectwrapped in moistened gauze. Automated robotic procedures involvingcutting blades, lasers and other methods of tissue separation are alsocontemplated. After these tissues are removed, the resulting ECMconsists mainly of epithelial basement membrane and subjacent tunicapropria.

In another embodiment, the ECM is prepared by abrading porcine bladdertissue to remove the outer layers including both the tunica serosa andthe tunica muscularis (layers G and F in FIG. 2) using a longitudinalwiping motion with a scalpel handle and moistened gauze. Followingeversion of the tissue segment, the luminal portion of the tunica mucosa(layer H in FIG. 2) is delaminated from the underlying tissue using thesame wiping motion. Care is taken to prevent perforation of thesubmucosa (layer E of FIG. 2). After these tissues are removed, theresulting ECM consists mainly of the tunica submucosa (layer E of FIG.2).

In another embodiment ECM is prepared as a powder. Such powder can bemade according the method of Gilbert et al., Biomaterials 26 (2005)1431-1435, herein incorporated by reference in its entirety. Forexample, UBM sheets can be lyophilized and then chopped into smallsheets for immersion in liquid nitrogen. The snap frozen material canthen be comminuted so that particles are small enough to be placed in arotary knife mill, where the ECM is powdered. Similarly, byprecipitating NaCl within the ECM tissue the material will fracture intouniformly sized particles, which can be snap frozen, lyophilized, andpowdered.

According to another embodiment, an extracellular matrix-derived gel isprovided. In certain embodiments, the method for making such a gelcomprises: (i) comminuting an extracellular matrix, (ii) solubilizingintact, non-dialyzed or non-cross-linked extracellular matrix bydigestion with an acid protease in an acidic solution to produce adigest solution, (iii) raising the pH of the digest solution to a pHbetween 7.2 and 7.8 to produce a neutralized digest solution, and (iv)gelling the solution at a temperature greater than approximately 25° C.The ECM typically is derived from mammalian tissue, such as, withoutlimitation from one of urinary bladder, spleen, liver, heart, pancreas,ovary, or small intestine. In certain embodiments, the ECM is derivedfrom a pig, cow, horse, monkey, or human. In one non-limitingembodiment, the ECM is lyophilized and comminuted. The ECM is thensolubilized with an acid protease. The acid protease may be, withoutlimitation, pepsin or trypsin, and in one embodiment is pepsin. The ECMtypically is solubilized at an acid pH suitable or optimal for theprotease, such as greater than about pH 2, or between pH 2 and 4, forexample in a 0.01M HCl solution. The solution typically is solubilizedfor 12-48 hours, depending upon the tissue type (e.g., see examplesbelow), with mixing (stirring, agitation, admixing, blending, rotating,tilting, etc.).

Once the ECM is solubilized the pH is raised to between 7.2 and 7.8, andaccording to one embodiment, to pH 7.4. Bases, such as bases containinghydroxyl ions, including NaOH, can be used to raise the pH of thesolution. Likewise buffers, such as an isotonic buffer, including,without limitation, Phosphate Buffered Saline (PBS), can be used tobring the solution to a target pH, or to aid in maintaining the pH andionic strength of the gel to target levels, such as physiological pH andionic conditions. The neutralized digest solution can be gelled attemperatures approaching 37° C., typically at any temperature over 25°C., though gelation proceeds much more rapidly at temperatures over 30°C., and as the temperature approaches physiological temperature. Themethod typically does not include a dialysis step prior to gelation,yielding a more-complete ECM-like matrix that typically gels at 37° C.more slowly than comparable collagen or dialyzed ECM preparations.

As described herein, the composition can be molded into any shape by anysuitable method, including, without limitation, placing into or onto amold, electrodeposition, and injection into a cavity or onto a surfacein a patient. Further, a molded gel can be trimmed and otherwise shapedby cutting or other suitable methods. In one non-limiting embodiment,the gel is injected into a site on a patient to add additional bulk orto fill in a void, for example, resulting from trauma or from removal ordegradation of tissue. In one non-limiting embodiment, the acidicsolubilization solution is mixed in a static mixer with a base and/orbuffer during injection into a patient. In further embodiments, cells,drugs, cytokines and/or growth factors can be added to the gel prior to,during or after gelation, so long as the bioactivity of the cells,drugs, cytokines and/or growth factors is not substantially orpractically (for the intended use) affected by the processing of the gelto its final form.

The ECM can be sterilized by any of a number of standard methods withoutloss of function. For example and without limitation, the material canbe sterilized by propylene oxide or ethylene oxide treatment, gammairradiation treatment (0.05 to 4 mRad), gas plasma sterilization,peracetic acid sterilization, or electron beam treatment. Treatment withglutaraldehyde results in sterilization as well as increasedcross-linking of the ECM. This treatment substantially alters thematerial such that it is slowly resorbed or not resorbed at all andincites a different type of host remodeling, which more closelyresembles scar tissue formation or encapsulation rather thanconstructive remodeling. If desired, cross-linking of the proteinmaterial within the ECM can also be induced with, for example andwithout limitation, carbodiimide isocyanate treatments, dehydrothermalmethods, and photooxidation methods. In one non-limiting embodiment, theECM is disinfected by immersion in 0.1% (v/v) peracetic acid, 4% (v/v)ethanol, and 96% (v/v) sterile water for two hours. The ECM material isthen washed twice for 15 minutes with PBS (pH=7.4) and twice for 15minutes with deionized water. The ECM-derived material may be furtherprocessed by optional drying, desiccation, lyophilization, freezedrying, and/or glassification. The ECM-derived material optionally canbe further digested, for example and without limitation by hydration (ifdried), acidification, enzymatic digests with, for example and withoutlimitation, trypsin or pepsin and neutralization.

Commercially available ECM preparations can also be used as thebiological polymeric component of the scaffold. In one non-limitingembodiment, the ECM is derived from small intestinal submucosa or SIS.Commercially available preparations include, but are not limited to,Surgisis™, Surgisis-ES™, Stratasis™, and Stratasis-ES™ (Cook UrologicalInc.; Indianapolis, Ind.) and GraftPatch™ (Organogenesis Inc.; CantonMass.). In another non-limiting embodiment, the ECM is derived fromdermis. Commercially available preparations include, but are not limitedto Pelvicol™ (sold as Permacol™ in Europe; Bard, Covington, Ga.),Repliform™ (Microvasive; Boston, Mass.) and Alloderm™ (LifeCell;Branchburg, N.J.). In another embodiment, the ECM is derived fromurinary bladder. Commercially available preparations include, but arenot limited to UBM (ACell Corporation; Jessup, Md.).

In general, the biodegradable scaffold described herein may be madeusing any useful method, including one of the many common processesknown in the polymer and textile arts. The biodegradable scaffold maytake many different forms. In certain non-limiting embodiments, thebiodegradable scaffold comprises a thin, flexible fabric that can besewn directly on to the site to be treated. In another non-limitingembodiment, the scaffold comprises a non-woven mat that can be suturedin place at the site of implantation or affixed using a medicallyacceptable adhesive. In one non-limiting embodiment, the scaffold issubstantially planar (having much greater dimension in two dimensionsand a substantially smaller dimension in a third, comparable tobandages, gauze, and other substantially flexible, flat items). Inanother non-limiting embodiment, the biodegradable scaffold comprises anon-woven fibrous article formed by electrodeposition of a suspensioncontaining the synthetic polymeric component and the biologicalpolymeric component. In yet another non-limiting embodiment, thebiodegradable scaffold comprises a porous composite formed by thermallyinduced phase separation.

The biodegradable scaffold can also have three-dimensional shapes usefulfor treating wounds and tissue deficiencies, such as plugs, rings,wires, cylinders, tubes, or disks. A useful range of thickness for thebiodegradable scaffold is between from about 10 μm (micrometers ormicrons (μ)) to about 3.5 cm, including increments therebetween,including, without limitation from about 10 μm to about 50 μm, 50 μm to3.5 cm, 100 μm to 3.0 cm, and between 300 μm and 2.5 cm.

In certain embodiments, the shape of the device is useful for replacingor repairing cartilagenous disc, such as, for example atemporomandibular joint meniscus. In further embodiments as shown inFIGS. 3 and 4, the disc replacement device 100 comprises anultrastructure similar to a hat wherein a central core 110 is thickwhile the surrounding brim 120 is thin. The advantage of the design isthat the central core 110 carries the compressive and shear forcesbetween the mandible and the skull, while the brim 120 allows the deviceanchored at specific points 130 (both laterally as well as medially),and, thus, attached to the surrounding tissue such that it remains inplace, but has sufficient movement to allow the mandible to move withoutgrinding against the skull. The brain may also serve as a conduit forthe migration and infiltration of cells into the acellular ECM scaffold.

Thus, three ultrastructural parameters operate in unison to allow thedevice to function even at the moment of implantation: the height andcomposition of the core which modulates compressive force (e.g., deformsunder stress), the diameter of circular core including the major andminor diameters where the core is elliptical which modulates the lateraldisplacement at the site of implantation, and the radius of the brimwhich modulates the type and number of attachment points for the devicein the implant site. Moreover, the functionality of the device is notdegraded over time, as the microstructure allows for influx of cells,thus allowing the body to remodel the device to the unique anatomy ofeach implanted individual.

As shown in FIGS. 5 and 6, such a device 200 can comprise a central core210 comprising particulate ECM powder or gel 215 which is encased inlayers of laminar ECM 230 and 240 that extend from the core to form abrim 220. The particulate ECM can be any suitable size including, forexample, particles with an average diameter of 10-400 μm, 1-500 μm,1-700 μm, or any other suitable diameter. In accordance, averageparticle diameters include those with diameters of 50 μm, 100 μm, 150μm, 200 μm, 250 μm and others include those with a diameter of 158 μmand 191 for example. Moreover the shape of the core can be concave,convex as shown in FIGS. 5 and 6, bi-concave (as in the original TMJmeniscus), bi-convex, or any combination thereof.

Independent of the particle size, in certain embodiments the devicecomprises a sheath which forms the brim as well as the encapsulation ofthe core. As such, the sheath can comprise one or more layers of laminarECM. For example, the device can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more layers. In certainembodiments, the layers themselves can substitute for the particulatematter. Thus, the disc itself can comprise 5, 10, 15, or 20 or morelayers of laminar ECM encapsulated in a sheath of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more layers.

Those of skill in the art recognize that the diameter of the core andthe width of the brim of a device described herein will depend on theanatomy of the individual. However, the height of the core can bebetween 0.5 and 4 cm, in certain instances. It is also contemplated thatthe core diameter can range from 8 to 12 mm, while the diameter of theentire device can range from 16 to 20 mm. The implant can bemanufactured in a range of sizes. For example in certain embodimentssmall, medium, and large implants can be available depending on thepatient's anatomy. For example and without limitation, an implant canhave a core diameter by total diameter of 8 mm×16 mm; 10 mm×18 mm; and12 mm×20 mm, respectively. The brim can be trimmed using scissors at thetime of surgery to provide an optimal fit. The physical properties ofthe device can be such that the size and shape change as a function ofthe mechanical loads placed upon the device during movement of theadjacent body parts.

The biodegradable scaffolds may be porous. Porosity may be accomplishedby a variety of methods. Although the biodegradable scaffolds may beporous or non-porous, it is often advantageous to use a process thatproduces a porous elastomeric scaffold. Non-limiting examples of suchprocesses include solvent casting/salt leaching, electrodeposition, andthermally induced phase separation. In other examples, porosity may beaccomplished by creating a mesh of fibers, such as by the aforementionedelectrodeposition or by any suitable method of producing a woven ornon-woven fiber matrix. As used herein, the term “porosity” refers to aratio between a volume of all the pores within the polymer compositionand a volume of the whole polymer composition. For instance, a polymercomposition with porosity of 85% would have 85% of its volume containingpores and 15% of its volume containing the polymer. In certainnon-limiting embodiments, the porosity of the scaffold is at least 60%,65%, 70%, 75%, 80%, 85%, or 90%, or increments therebetween. In anothernon-limiting embodiment, the average pore size of the scaffold isbetween 0.1 and 300 microns, including increments therebetween. Forexample and without limitation, a biodegradable scaffold that acts as abarrier to bacteria and other pathogens may have an average pore size ofless than 0.5 microns or less than 0.2 microns. When the scaffold is tobe manufactured by electrodeposition, it is often advantageous to adjustthe pore size or degree of porosity by varying the polymer concentrationof the electrodeposition solution or by varying the spinning distancefrom the nozzle to the target. For example and without limitation, theaverage pore size may be increased by increasing the amount of polymericcomponents within the suspension used for electrodeposition, whichresults in larger fiber diameters and therefore larger pore sizes. Inanother non-limiting example, the average pore size can be increased byincreasing spinning distance from the nozzle to the target, whichresults in less adherence between fibers and a looser matrix.

In certain non-limiting embodiments, the biodegradable scaffold is madeby using solvent casting and salt leaching. This method involvesdissolving the polymeric components that constitute the scaffold into asuitable organic solvent and then casting the solution into a moldcontaining small particles of predetermined size (known as porogens).Examples of suitable porogens include inorganic salts, crystals ofsaccharose, gelatin spheres or paraffin spheres. By adjusting theporogen size and/or the ratio of porogen to solvent, the porosity of thefinal elastomeric scaffold may be adjusted. After casting, the solventis evaporated, and the resulting polymer composition is immersed into asecond solvent that dissolves the porogen, but not the polymer, toproduce a porous, sheet-like structure.

In other non-limiting embodiments, electrodeposition is used tofabricate the scaffold. The process of electrodeposition involvesplacing a polymer-containing fluid (for example, a polymer solution, apolymer suspension, or a polymer melt) in a reservoir equipped with asmall orifice, such as a needle or pipette tip and a metering pump. Oneelectrode of a high voltage source is also placed in electrical contactwith the polymer-containing fluid or orifice, while the other electrodeis placed in electrical contact with a target (typically a collectorscreen or rotating mandrel). During electrodeposition, thepolymer-containing fluid is charged by the application of high voltageto the solution or orifice (for example, about 3-15 kV) and then forcedthrough the small orifice by the metering pump that provides steadyflow. While the polymer-containing fluid at the orifice normally wouldhave a hemispherical shape due to surface tension, the application ofthe high voltage causes the otherwise hemispherically shapedpolymer-containing fluid at the orifice to elongate to form a conicalshape known as a Taylor cone. With sufficiently high voltage applied tothe polymer-containing fluid and/or orifice, the repulsive electrostaticforce of the charged polymer-containing fluid overcomes the surfacetension and a charged jet of fluid is ejected from the tip of the Taylorcone and accelerated towards the target, which typically is biasedbetween −2 to −10 kV. Optionally, a focusing ring with an applied bias(for example, 1-10 kV) can be used to direct the trajectory of thecharged jet of polymer-containing fluid. As the charged jet of fluidtravels towards the biased target, it undergoes a complicated whippingand bending motion. If the fluid is a polymer solution or suspension,the solvent typically evaporates during mid-flight, leaving behind apolymer fiber on the biased target. If the fluid is a polymer melt, themolten polymer cools and solidifies in mid-flight and is collected as apolymer fiber on the biased target. As the polymer fibers accumulate onthe biased target, a non-woven, porous mesh is formed on the biasedtarget.

The properties of the electrodeposited scaffolds can be tailored byvarying the electrodeposition conditions. For example, when the biasedtarget is relatively close to the orifice, the resultingelectrodeposited mesh tends to contain unevenly thick fibers, such thatsome areas of the fiber have a “bead-like” appearance. However, as thebiased target is moved further away from the orifice, the fibers of thenon-woven mesh tend to be more uniform in thickness. Moreover, thebiased target can be moved relative to the orifice. In certainnon-limiting embodiments, the biased target is moved back and forth in aregular, periodic fashion, such that fibers of the non-woven mesh aresubstantially parallel to each other. When this is the case, theresulting non-woven mesh may have a higher resistance to strain in thedirection parallel to the fibers, compared to the directionperpendicular to the fibers. In other non-limiting embodiments, thebiased target is moved randomly relative to the orifice, so that theresistance to strain in the plane of the non-woven mesh is isotropic.The properties of the electrodeposited elastomeric scaffold may also bevaried by changing the magnitude of the voltages applied to theelectrodeposition system. In one non-limiting embodiment, theelectrodeposition apparatus includes an orifice biased to 12 kV, atarget biased to −7 kV, and a focusing ring biased to 3 kV. Moreover, auseful orifice diameter is 0.047″ (I.D.) and a useful target distance isabout 23 cm. Other electrodeposition conditions that can be variedinclude, for example and without limitation, the feed rate of thepolymer solutions, the solution concentrations, and the polymermolecular weight. Non-limiting examples of useful range of high-voltageto be applied to the polymer suspension is from 0.5 to 30 kV, from 5 to25 kV, and from 10 to 15 kV.

In another non-limiting embodiment, thermally induced phase separation(TIPS) is used to fabricate the biodegradable elastomeric scaffold. Thismethod involves dispersing the polymeric components in a solvent (forexample and without limitation, DMSO—dimethyl sulfoxide) and thencasting, for example by injecting or otherwise placing the compositioninto a mold. The mold can have any useful shape, such as a sheet or net.In a typical TIPS fabrication process, a pre-formed mold is cooled tolow temperature (for example and without limitation −80° C.), whichcauses the polymeric components to separate out of the solvent. The moldis then transferred to ethanol to extract the DMSO.

Fabrication and modification of the scaffold can comprise multiple stepsusing multiple techniques using polymer compositions that are the sameor different. In one non-limiting example, TIPS is used to fabricate thescaffold and electrodeposition is used to form a fiber coating onto oraround the scaffold. In another non-limiting example, solventcasting/salt leaching is used to fabricate the scaffold andelectrodeposition is used to form a fiber coating onto or around thescaffold. The electrodeposition solution can contain one or more of anypolymeric components, including synthetic polymeric components,biological polymeric components, or mixtures of both. The fiber coatingformed by electrodeposition can be coated onto or around the entirescaffold or portions of the scaffold.

After fabricating the scaffold, the planar or three-dimensional surfaceof the scaffold may be functionally modified (functionalized) for anypurpose, such as, without limitation, to promote cellular adhesion andmigration onto and/or into the scaffold. In one non-limiting example,the surface is first treated to introduce a reactive group on thesurface by any useful process, such as one of the many processes knownin the art. Second, the activated surface is reacted with anadhesion-promoting peptide or group. The reactive group on the surfacecan be, for example and without limitation, a hydroxyl group or an aminegroup. In one embodiment, radio-frequency glow discharge is used toproduce plasma containing ammonia gas and amine groups are introduced tothe surface by treatment with the plasma. In another embodiment,radio-frequency glow discharge is used to introduce hydroxyl groups tothe surface by treatment with plasma.

The activated surface can be modified with an adhesion-promotingoligopeptide to promote cellular ingrowth into and/or onto the scaffold.Non-limiting examples of adhesion-promoting oligopeptides include: RGDor RGDS (SEQ ID NO.: 1), a recognition site for fibronectin,vitronectin, fibrinogen, von Willebrand factor, and collagen; LDV, REDV(SEQ ID NO.: 2), PHSRN (SEQ ID NO.: 3), and KNEED (SEQ ID NO.: 4), whichare recognition sites for fibronectin; YIGSR (SEQ ID NO.: 5) and IKVAV(SEQ ID NO.: 6), which are recognition sites for laminin; and DGEA (SEQID NO.: 7), a recognition site for collagen.

In one specific non-limiting embodiment, the scaffold is functionalizedto present the peptide RGDS (SEQ ID NO.: 1) on its surface. First, thesurface is treated with radio-frequency glow discharge containingammonia gas to introduce amine groups. Ammonia-containing gas isgenerated by connecting a flask containing ammonium hydroxide (30 wt %solution) to the glow discharge reactor and maintaining pressure at3×10⁻³ Torr. The surface is further treated with 1,4-diisocyanatobutaneto provide a reactive isocyanate group. Next, RGDS (SEQ ID NO.: 1) isattached to the activated surface. The activated surface is immersed ina solution of 20 μg/mL RGDS (SEQ ID NO.: 1) in PBS for 10 hours and thenrinsed with PBS.

One or more of therapeutic agents can be introduced into the scaffold byany useful method, such as, without limitation absorption, adsorption,deposition, admixture with a polymer composition used to manufacture thescaffold and linkage of the agent to a component of the scaffold. In onenon-limiting example, the therapeutic agent is introduced into abackbone of a polymer used in the scaffold. By adding the therapeuticagent to the polymer itself, the rate of release of the therapeuticagent may be controlled by the rate of polymer degradation. In anothernon-limiting example, the therapeutic agent is introduced when thescaffold is being made. For instance, during a solvent casting or TIPSprocess, the therapeutic agent can be added to the solvent with thepolymer in the pre-formed mold. During an electrodeposition process, thetherapeutic agent can be electrosprayed onto the polymer being spun. Inyet another non-limiting example, the therapeutic agent is introducedinto the scaffold after the device is made. For instance, the scaffoldmay be “loaded” with therapeutic agent(s) by using static methods. Forinstance, the scaffold can be immersed into a solution containing thetherapeutic agent, permitting the agent to absorb into and/or adsorbonto the scaffold. The scaffold may also be loaded by using dynamicmethods. For instance, a solution containing the therapeutic agent canbe perfused or electrodeposited into the scaffold. In another instance,a therapeutic agent can be added to the scaffold before it is implantedin the patient.

Therapeutic agents within the scaffold can be used in any number ofways. In one non-limiting embodiment, a therapeutic agent is releasedfrom the scaffold. For example and without limitation, anti-inflammatorydrugs are released from the scaffold to decrease an immune response. Inanother non-limiting embodiment, a therapeutic agent is intended tosubstantially remain within the scaffold. For example and withoutlimitation, chemoattractants are maintained within the scaffold topromote cellular migration and/or cellular infiltration into thescaffold.

In one non-limiting embodiment, the scaffolds release therapeutic agentswhen the polymeric components degrade within the patient's body. Forexample and without limitation, the individual building blocks of thepolymers may be chosen such that the building blocks themselves providea therapeutic benefit when released in situ through the degradationprocess. In one non-limiting embodiment, one of the polymer buildingblocks is putrescine, which has been implicated as a substance thatcauses cell growth and cell differentiation.

In another non-limiting embodiment, at least one therapeutic agent isadded to the scaffold before it is implanted in the patient. Generally,the therapeutic agents include any substance that can be coated on,embedded into, absorbed into, adsorbed onto, or otherwise attached to orincorporated onto or into the scaffold that would provide a therapeuticbenefit to a patient. Non-limiting examples of such therapeutic agentsinclude antimicrobial agents, growth factors, emollients, retinoids, andtopical steroids. Each therapeutic agent may be used alone or incombination with other therapeutic agents. For example and withoutlimitation, a scaffold comprising neurotrophic agents or cells thatexpress neurotrophic agents may be applied to a wound that is near acritical region of the central nervous system, such as the spine.Alternatively, the therapeutic agent may be blended with the polymerwhile the polymer is being processed. For example, the therapeutic agentmay be dissolved in a solvent (e.g., DMSO) and added to the polymerblend during processing. In another embodiment, the therapeutic agent ismixed with a carrier polymer (e.g., polylactic-glycolic acidmicroparticles) which is subsequently processed with an elastomericpolymer. By blending the therapeutic agent with a carrier polymer orelastomeric polymer itself, the rate of release of the therapeutic agentmay be controlled by the rate of polymer degradation.

In certain non-limiting embodiments, the therapeutic agent is a growthfactor, such as a neurotrophic or angiogenic factor, which optionallymay be prepared using recombinant techniques. Non-limiting examples ofgrowth factors include basic fibroblast growth factor (bFGF), acidicfibroblast growth factor (aFGF), vascular endothelial growth factor(VEGF), hepatocyte growth factor (HGF), insulin-like growth factors 1and 2 (IGF-1 and IGF-2), platelet derived growth factor (PDGF), stromalderived factor 1 alpha (SDF-1 alpha), nerve growth factor (NGF), ciliaryneurotrophic factor (CNTF), neurotrophin-3, neurotrophin-4,neurotrophin-5, pleiotrophin protein (neurite growth-promoting factor1), midkine protein (neurite growth-promoting factor 2), brain-derivedneurotrophic factor (BDNF), tumor angiogenesis factor (TAF),corticotrophin releasing factor (CRF), transforming growth factors ccand β (TGF-α and TGF-β), interleukin-8 (IL-8), granulocyte-macrophagecolony stimulating factor (GM-CSF), interleukins, and interferons.Commercial preparations of various growth factors, includingneurotrophic and angiogenic factors, are available from R & D Systems,Minneapolis, Minn.; Biovision, Inc, Mountain View, Calif.; ProSpec-TanyTechnoGene Ltd., Rehovot, Israel; and Cell Sciences®, Canton, Mass.

Methods of promoting wound healing or tissue generation or regenerationin a patient also are provided. The methods comprise, withoutlimitation, implanting a scaffold as described herein at or near a sitefor wound healing or tissue generation or regeneration in the patient.In any such method, the scaffold may comprise a therapeutic agent asdescribed herein.

In certain non-limiting embodiments, the therapeutic agent is anantimicrobial agent, such as, without limitation, isoniazid, ethambutol,pyrazinamide, streptomycin, clofazimine, rifabutin, fluoroquinolones,ofloxacin, sparfloxacin, rifampin, azithromycin, clarithromycin,dapsone, tetracycline, erythromycin, ciprofloxacin, doxycycline,ampicillin, amphotericin B, ketoconazole, fluconazole, pyrimethamine,sulfadiazine, clindamycin, lincomycin, pentamidine, atovaquone,paromomycin, diclazaril, acyclovir, trifluorouridine, foscarnet,penicillin, gentamicin, ganciclovir, iatroconazole, miconazole,Zn-pyrithione, and silver salts such as chloride, bromide, iodide andperiodate.

In certain non-limiting embodiments, the therapeutic agent is ananti-inflammatory agent, such as, without limitation, a NSAID, such assalicylic acid, indomethacin, sodium indomethacin trihydrate,salicylamide, naproxen, colchicine, fenoprofen, sulindac, diflunisal,diclofenac, indoprofen, sodium salicylamide; an anti-inflammatorycytokine; an anti-inflammatory protein; a steroidal anti-inflammatoryagent; or an anti-clotting agents, such as heparin. Other drugs that maypromote wound healing and/or tissue regeneration may also be included.

In certain non-limiting embodiments, the therapeutic agent comprisescells that are added to the scaffold before or at the time ofimplantation. In such embodiments, it is often advantageous to use aporous biodegradable elastomeric scaffold, so that the cells may beincorporated into the porous structure of the scaffold (a conditionreferred to as “microintegration”). In this way, most of the cells willhave a tendency to be trapped or otherwise contained within the porousstructure of the scaffold. The cells that are microintegrated may remainafter the scaffold has fully disintegrated within the patient. However,the microintegrated cells may also be merely cells that act asprecursors to the final tissue that is formed when the scaffold hasfully degraded.

Cells may be autologous (obtained from the patient to receive thescaffold), from an allogeneic or xenogeneic source or from any usefulcell line, such as, without limitation, stem cells or precursor cells(cells that can differentiate into another cell type) that are capableof cellular growth, remodeling, and/or differentiation. By way ofexample only, the cells that may be incorporated onto or into thescaffold include stem cells, precursor cells, smooth muscle cells,skeletal myoblasts, myocardial cells, endothelial cells, fibroblasts,chondrocytes and genetically modified cells. Various commerciallyavailable cell lines include Clonetics® Primary Cell Systems (LonzaGroup, Inc., Switzerland), ATCC.

Cells may be microintegrated with the scaffold using a variety ofmethods. For example and without limitation, the scaffold may besubmersed in an appropriate growth medium for the cells of interest, andthen exposed to the cells. The cells are allowed to proliferate on thesurface and interstices of the scaffold. The scaffold is then removedfrom the growth medium, washed if necessary, and implanted.Alternatively, the cells may be placed in a suitable buffer or liquidgrowth medium and drawn through the scaffold by using vacuum filtration.In another non-limiting embodiment, the cells of interest are dissolvedinto an appropriate solution (e.g., a growth medium or buffer) and thensprayed onto a scaffold while the scaffold is being formed byelectrodeposition. In yet another non-limiting embodiment, the cells areplaced in a solution that is biased and then electrosprayed onto thescaffold while it is being electrodeposited. By way of example only, thecells that may be incorporated on or into the scaffold includechondrocytes, stem cells, precursor cells, smooth muscle cells, skeletalmyoblasts, myocardial cells, endothelial cells, fibroblasts andgenetically modified cells.

In one non-limiting embodiment, the genetically modified cells arecapable of expressing a therapeutic substance, such as a growth factor.Cells can be modified by any useful method in the art. For example andwithout limitation, the therapeutic agent is a growth factor that isreleased by cells transfected with cDNA encoding for the growth factor.Therapeutic agents that can be released from cells include, withoutlimitation, a neurotrophic factor, such as nerve growth factor,brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4,neurotrophin-5, and ciliary neurotrophic factor; a growth factor, suchas basic fibroblast growth factor (bFGF), acidic fibroblast growthfactor (aFGF), vascular endothelial growth factor (VEGF), hepatocytegrowth factor (HGF), insulin-like growth factors (IGF), platelet derivedgrowth factor (PDGF), transforming growth factor-beta (TGF-β),pleiotrophin protein (neurite growth-promoting factor 1), and midkineprotein (neurite growth-promoting factor 2); an anti-inflammatorycytokine; and an anti-inflammatory protein. The cells may be autologous,allogeneic, etc.

In addition to providing scaffolds as described above, methods of usingsuch elastomeric scaffolds are encompassed herein. Generally, a scaffoldcan be implanted by using any suitable medical procedure thatfacilitates use of the scaffold to provide a therapeutic benefit. Asused herein, the terms “implanted” and “implantation” and like termsrefer to an act of delivering a scaffold or scaffold-containing deviceto a site within the patient and of affixing the scaffold or device tothe site. The site of implantation in a patient typically is “at or neara site for wound healing or tissue generation or regeneration in thepatient,” meaning the scaffold-containing device is implanted in, on,onto, adjacent to or in proximity to a desired site of delivery tofacilitate healing and/or tissue generation or regeneration to repair aninjury or defect in the patient and/or to achieve a desired effect inthe patient, such as wound drainage. The delivery method may alsoinclude minimally invasive methods such as by catheter based technologyor by needle injection. The patient may be human or animal. The scaffoldmay be delivered by any surgical procedure, including minimally invasivetechniques, such as laparoscopic surgery, as well as invasive techniquessuch as thoracic surgery and fasciotomy. The scaffold or device may beimplanted alone or implanted in conjunction with surgical fasteners,such as sutures, staples, adhesives, screws, pins, and the like.Additionally, biocompatible adhesives, such as, without limitation,fibrin-based glue) may be used to fasten the scaffolds as well.

In yet another non-limiting embodiment, the scaffold can be in the formof a powder or fine particles (for example, formed by shredding anon-woven mesh formed by electrodeposition or TIPS). In thesesituations, it may be advantageous to derivatize the elastomericscaffold with therapeutic agents, such as antibiotics or growth factors,prior to insertion into the wound.

Example 1 Preparation of Powdered Extracellular Matrix from PorcineUrinary Bladders (UBM)

Porcine urinary bladders were harvested from pigs immediately followingeuthanasia. Connective tissue and adipose tissue were removed from theserosal surface and any residual urine was removed by repeated washeswith tap water. The tunica serosa, tunica mucosa externa, the tunicasubmucosa, and most of the tunica muscularis interna were mechanicallyremoved and the luminal urothelial cells of the basement membrane weredissociated by soaking in 1.0 N saline solution yielding a biomaterialcomposed primarily or exclusively of the basement membrane plus thesubjacent tunica propria. This bi-laminate structure was referred to asurinary bladder matrix (UBM). UBM sheets were disinfected for two hourson a shaker in a solution containing 0.1% (v/v) peracetic acid, 4% (v/v)ethanol, and 95.9% (v/v) sterile water. The peracetic acid residue wasremoved by washing with sterile phosphate-buffered saline (pH=7.4) twicefor 15 minutes each and twice for 15 minutes each with sterile water.UBM sheets were lyophilized and then chopped into small sheets forimmersion in liquid nitrogen. The snap frozen material was then reducedto small pieces with a WARING™ blender so that the particles were smallenough to be placed in a rotary knife mill. A #60 screen was used torestrict the collected powder size to less than 250 mm. Sonic siftingand laser diffraction were used to analyze the particle sizedistribution that resulted from the powdering methods as described inGilbert et al., Biomaterials 26 (2005) 1431-1435, herein incorporated byreference in its entirety. The particles formed were irregularly shapedand could be defined generally as sheet-like or fiber-like.

In another method, the ECM was first soaked in the disinfected materialin a 30% (w/v) NaCl solution for 5 min. The material was then snapfrozen in liquid nitrogen to precipitate salt crystals, and lyophilizedto remove residual water. This material was then comminuted as describedsupra. By precipitating NaCl within the tissue, it was expected that theembedded salt crystals would cause the material to fracture into moreuniformly sized particles. The particles were suspended in deionizedwater and centrifuged for 5 min at 1000 rpm three times to remove theNaCl. The suspension was snap frozen and lyophilized again. Finally, thepowder was placed in a rotary knife mill to disaggregate the individualparticles. Sonic sifting and laser diffraction were used to analyze theparticle size distribution that resulted from the two powdering methods.Sonic sifting involved separating the powder by size through a series ofgraduated screens stacked in a vertical configuration. The powder passedthrough the screens as a result of sonic pulses along the longitudinalaxis of the stack and mechanical agitation in the plane of the screens.The screen sizes used were 212, 125, 90, 63, and 38 mm. The mass of thepowder from each cut was weighed and the amount of material in each cutwas represented as the percentage of the total mass. This data wasconverted to a percentage of the total volume based on an assumptionthat the density of the material was the same for the two productionmethods.

The ultrastructure for particles produced by the first method wasevaluated with SEM. The particles formed by the first method wereirregularly shaped and could be defined generally as sheet-like orfiber-like. Examination of the particles at higher magnification (500×)showed that two distinct surface ultrastructures were present. Onesurface appeared to be quite smooth while the other appeared to be morefibrous. Since sonic sifting did not give an accurate description of theparticle size distribution, only laser diffraction was used to analyzethe size of the UBM powder produced by the salt precipitation method.The mean particle size was found to be smaller for powder produced bythe salt precipitation method than the particle size produced by thefirst method. Furthermore, the size of the particles was more uniformwhen produced by the salt precipitation method. The particles formedafter salt precipitation showed a different morphology than those formedby the first method as shown with SEM. The larger particles in thedistribution (150-200 μm) appeared to have porous surfaces and there wasa large population of very fine particles (approximately 1 μm) whichtended to associate with the larger particles or agglomerate.

Example 2 Manufacture of a TMJ Meniscus Replacement Device

Laminar ECM was layered into a pressure mold device. Urinary bladdermatrix (UBM) sheets were prepared as described in Example 1 above. Afterundergoing a decellularization step, hydrated sheets of UBM were cut andplaced, with the luminal surface facing the mold, onto a pressure molddevice. In various embodiments, anywhere from 1 to about 20 layers ofUBM were used to create the device. The mold consisted of a block ofhard plastic that has been milled to create a depression in the centerwith the approximate size of the desired central core (see e.g., 110 ofFIGS. 3 and 4) of the TMJ device.

Non-limiting examples of molds include FIG. 9, which shows a schematicof a mold for a canine TMJ meniscus replacement device, and FIG. 10,which shows a mold for a human TMJ meniscus replacement device.Dimensions are shown in millimeters (mm).

Following placement of the sheet form of the UBM (or any other ECM), thesheets are pressed such that they will line the inside of thedepression, creating a pocket where the particulate ECM (or gel, ormultiple layers of the sheet form) will be placed. Particulate ECMpowder manufactured as in Example 1 was then used to fill the void inthe mold. Notably, any particulate or gel or sheets could be packed intothe pocket until the desired fill is achieved (e.g., ˜200 mg of powderin the canine devices). Additional layers of laminar ECM were thenplaced over the mold.

More ECM sheets were then cut to size and placed over top of thepreviously placed sheets and powder core to create an enclosed core ofECM powder. Again, the number of sheets placed can range from 1 to about20 layers. The mold, sheet, powder, sheet construct were then coveredwith cheese cloth and a piece of metal mesh and sealed inside of aplastic pouch for vacuum pressing. The device was then pressed using avacuum pump with a condensate trap inline. The constructs were subjectedto a vacuum of at least 28 inches Hg until dried, leaving amultilaminate construct. (See FIG. 7).

Example 3 Canine Model for TMJ Meniscus Implantation

Five female mongrel dogs of approximately 15-20 kg (purchased fromMarshall Bio-Resources USA (North Rose, N.Y.)) were subjected tounilateral meniscectomy followed by replacement of the meniscus with aUBM-ECM device. The UBM-ECM device consisted of particulate UBM-ECMencapsulated between sheets of UBM-ECM forming a “pillow-like” device.At time points of 3 weeks, 1, 2, 3, and 6 months, one animal wassacrificed and the condylar head, temporal fossa, and the UBM-ECMimplant were excised and assessed using histologic andimmunohistochemical methods.

UBM-ECM Device Preparation

UBM-ECM was prepared from porcine bladders. Briefly, urinary bladderswere harvested from market weight pigs immediately following sacrifice.The bladder tissue was rinsed in water to facilitate the removal ofexcess urine and the urothelial cell layer. Excess connective andadipose tissue was removed from the serosal surface of the bladder usingscissors. The apex of the bladder was removed and the bladder was thensplit longitudinally from the apical opening to the neck of the bladderforming a rectangular sheet. The tunica serosa, tunica muscularisexterna, tunica submucosa, and the majority of the muscularis mucosawere removed by mechanical delamination of the abluminal side of thebladder. The remaining tissue consisted of the basement membrane, tunicapropria, and resident cells.

The tissue was then treated in a 0.1% peracetic acid/4% ethanol solutionfor two hours to initiate decellularization and disinfect the tissue.Following treatment in the peracetic acid/ethanol solution, the tissuewas repeatedly washed in phosphate buffered saline (PBS) and water toremove cellular remnants and traces of the peracetic acid/ethanolsolution and to return the pH of the material to 7.4. The remainingdecellularized tissue was stored in water until used and represented thehydrated sheet form of UBM-ECM. A portion of the hydrated sheet form ofUBM-ECM was frozen and lyophilized. The dry sheet was cut into smallerpieces and comminuted using a Wiley mill with a #60 mesh screen. Thecomminuted UBM-ECM represented the particulate form of UBM-ECM.

A hard plastic mold was milled to create an oval-shaped depression withthe approximate size of the desired central core of the TMJ device (10mm×14 mm oval, 2 mm depth) and a flat surface surrounding the depressionto allow for the formation of a “pillow-like” core and a flat anchoringsite. Two hydrated sheets of UBM-ECM were then cut to size and placedonto the mold. Following placement, the hydrated sheets were pressed toline the inside of the depression, creating a pocket into whichparticulate UBM-ECM was packed. Approximately 200-300 mg of the ECMpowder was packed into the depression and two hydrated sheets were cutto size and placed over the top of the powder to create an enclosedcore. The constructs were subjected to a vacuum of at least 28 inches Hguntil dry, forming a multilaminate construct. All constructs wereterminally sterilized using ethylene oxide prior to implantation.

Surgical Procedure

All animals were sedated with acepromazine (01-0.5 mg/kg body weight)prior to intubation and maintenance of a surgical plane of anesthesiawith isoflurane (1-5%). The surgical site was shaved and prepared usinga betadine scrub prior to the placement of sterile drapes. An incisionwas made anterior to the tragus, preserving local innervation andvasculature. The native meniscus was then isolated and completelyremoved.

The UBM-ECM implants were hydrated in sterile saline for approximately10 minutes prior to use as a replacement device for the native meniscus.Devices were placed such that the powder “pillow” was situated betweenthe temporal fossa and the condylar head. Three holes were created inthe temporal fossa and the implants were then secured to the temporalfossa with fixation sutures. Fixation sutures were also placed in theanterior and posterior aspects of the implant through the adjacenttissues, and soft tissue and the skin was then closed using resorbablesuture material.

Post Operative Care

Following the surgical procedure, the animals were recovered fromanesthesia, extubated and monitored until resting comfortably in asternal position. The animals were then monitored and the followingparameters were recorded every 3 hours for the first 24 hours postsurgery: pulse rate, strength of pulse, capillary refill time,respiratory rate and ability to maintain an open airway, urinary output,and defecation. Body temperature was measured and recorded every 12hours. The animals were restricted to confinement housing (not more than2-3 days) until stable, and were then placed in 10×14 ft runs andallowed free movement. Buprenorphine was administered (0.005-0.01 mg/kgbody weight) for 5 days post operatively and then as needed thereafterfor pain management. The dogs were also given Cephalexin (35 mg/kg bodyweight) for 5 days post-operatively. Animals were fed a soft diet forthe first 5-7 days post operatively and were returned to a normal harddiet thereafter.

Euthanasia and Sample Harvest

At the predetermined date of sacrifice, the animals were sedated withacepromazine (0.1-0.5 mg/kg body weight), anesthetized using isoflurane(5%) and euthanized by intravenous administration of pentobarbitalsodium (390 mg/4.5 kg body weight). Following euthanasia, the temporalfossa, the condylar head, and the interpositional material between thestructures were excised and fixed in 10% neutral buffered formalin forhistologic and immunolabeling examination. Native fossa, condyle, andmeniscus tissues were also harvested as controls and were treated inidentical fashion as the experimental explant tissue.

Gross Morphologic Examination

At the time of explant, the joint space of all animals was examined forsigns of pathologic degeneration of the articulating surfaces of thetemporal fossa and mandibular condyle and/or other signs of pathologicjoint inflammation.

Histologic Evaluation

Formalin fixed tissues were embedded in paraffin, cut into 6 μm sectionsand mounted on glass slides. Sections were deparaffinized by immersionin xylenes followed by a graded series of ethanol. The slides werestained using hematoxylin and eosin or Herovici's polychrome, and werethen dehydrated in ethanol and xylenes prior to coverslipping. Theslides were then evaluated under light microscopy. Pre-implant UBM-ECMdevices were also subjected to histologic evaluation using the methodsdescribed above. Additionally, pre-implant UBM-ECM scaffolds werestained with 4′,6-diamidino-2-phenylindole (DAPI) to confirm completedecellularization.

Immunolabeling Studies

Sections of the TMJ meniscus tissue were labeled using antibodiesspecific for CD31 and CD68 to determine the presence of blood vesselsand macrophages, respectively, within the remodeling UBM-ECM implant.Slides were deparaffinized by immersion in xylenes and a graded seriesof ethanol. Antigen retrieval was then performed by boiling slides in 10mM citric acid monohydrate (pH 6.0) for 20 minutes. Following antigenretrieval, the slides were exposed to a solution consisting of TRISbuffered saline and 0.05% Tween 20. Slides were then washed in PBS threetimes for a total of 10 minutes and a solution of 3% H₂O₂ in methanolwas applied for 30 minutes at room temperature to quench endogenousperoxidase activity. Slides were blocked in a solution consisting of 2%normal serum, 1% BSA, 0.1% Triton-X 100, and 0.05% Tween 20 in PBS for30 minutes at room temperature. Primary antibodies were diluted in theblocking solution (1:250) and applied to the slides overnight at 4° C.Slides were washed in PBS, and secondary antibodies diluted in blockingsolution were applied for 30 minutes at room temperature. Slides werewashed in PBS and ABC reagent (Vector, Burlingame, Calif.) was appliedto for 30 minutes at 37° C. The slides were washed in PBS and waterprior to development using 4% diaminobenzadine substrate (Vector)solution. Finally, slides were counterstained using hematoxylin(Vector), dehydrated using the reverse of the dewaxing procedure above,and coverslipped for examination under light microscopy.

Results

All of the animals in this study survived the surgical procedure andlived until their predetermined sacrifice date without complication.

Gross Morphologic Findings

Gross morphologic examination showed that there was little to no changein the articulating surfaces of the temporal fossa or the mandibularcondyle at any time point following placement of the UBM-ECM device.There were no signs of synovitis, or excess fluid in the joint space.The UBM-ECM device showed progressive remodeling and was replaced with astructure that highly resembled the fibrocartilage of the native TMJdisk by the 6 month post surgical time point. It was not possible todifferentiate the original UBM-ECM device from newly deposited hosttissue at any of the time points investigated in this study.

Histopathologic Findings

Histologic evaluation was performed both at the center of the remodelingimplant and at the periphery of the implant to determine both the bulkmorphology of the remodeling device and the degree of integration of thedevice with the muscular tissues at the peripheral attachment sites.

Pre-Implantation UBM-ECM Device

Histologic staining of the UBM-ECM device showed small particles ofmature, well-organized collagenous extracellular matrix (UBM-ECM powder)encapsulated within sheets of the same. The particles were randomlyoriented and the internal structure of the device was highly porous. Theexterior surface of the device was also observed to be composed ofmature, well-organized collagenous extracellular matrix and wascharacterized by its smooth, dense structure (UBM-ECM sheets).

Remodeled UBM-ECM Device

Bulk Morphology

At three weeks the implanted UBM-ECM device was no longer identifiableand the site of remodeling was characterized by a dense infiltration ofpredominantly mononuclear cells within newly deposited ECM. Herovicistaining showed that the newly deposited ECM was composed of bothcollagen type I and small amounts of collagen type III.

At one month the site of remodeling was characterized by a dense,randomly distributed cellular infiltrate consisting of both mononuclearcells and spindle shaped cells. There was a decrease in the number ofmononuclear cells compared to the three week time point. Herovicistaining indicated that the remodeling site contained both collagen typeI and small amounts of collagen type III with an increase in both thedensity and the degree of organization of the newly deposited collagentype I.

At two months the site of remodeling was characterized by an increase inthe number of spindle shaped cells within the remodeling site with aconcomitant decrease in the number of mononuclear cells and a decreasein overall cellular density compared to earlier time points. Herovicistaining indicated that the remodeling site contained both collagen typeI and collagen type III with a predominance of collagen type I. It wasalso noted that the organization of the collagen was increased with amorphology that more resembled the native TMJ than did the collagendeposited in the remodeling site at the three week or one month timepoints.

At three months the density of the cellular infiltrate within the siteof remodeling was greatly decreased compared to all earlier time points.The cellular population at the three month time point was characterizedby predominantly spindle shaped cells with a small number of randomlydistributed mononuclear cells remaining within the remodeling site.Herovici staining showed that, as at previous time points, theremodeling site was characterized by a deposition of both collagen typeI and collagen type III with a predominance of collagen type I at thethree month time point. The density of the collagen type I deposited inthe remodeling site was greater than any of the previous time points,and the morphology of the collagen matrix present in the remodeling siteat three months highly resembled that of the native TMJ.

At six months the remodeling site was characterized by a sparsepopulation of spindle shaped cells within an aligned matrix ofcollagenous tissue. Herovici staining indicated that there were highlyorganized collagen type I fibers formed within the remodeling site withinterspersed collagen type III fibrils. The morphology of the remodeledtissue at 6 months was almost indistinguishable from that of the nativeTMJ.

B. Peripheral Musculature Attachment Site

Histologic staining showed that the newly deposited ECM within the siteof remodeling was well integrated with the native musculature at theperiphery of the implanted device as early as one monthpost-implantation. A dense population of small, dark staining cells wasobserved adjacent to bundles of skeletal muscle at the interface betweenthe remodeling UBM-ECM device and native host tissue at one month postimplantation. The degree of integration of the UBM-ECM device with thenative host tissue was shown to increase with time and ingrowth of hostskeletal muscle tissue into the site of remodeling was observed. By sixmonths post-implantation, bundles of skeletal muscle were observedwithin the site of remodeling and were surrounded by mature,well-organized collagenous extracellular matrix. Muscular ingrowth wasobserved only at the periphery, and not in the bulk, of the remodelingUBM-ECM device.

Articulating Surfaces

Macroscopic and histologic examination showed that there were nopathologic changes in the articulating surfaces of the condyle or thefossa at any of the time points investigated in this study. That is, thearticulating surfaces of the fossa and condyle were characterized by thepresence of smooth, thin fibrocartilagenous tissue which resembled thatobserved in the contralateral control at all of the time points examinedin this study.

Immunolabeling Findings

CD31

Immunolabeling for CD31 showed that there were a large number ofrandomly distributed blood vessels within the site of remodeling atearly time points (3 weeks and 1 month). Both the number and size of thevessels were shown to decrease by 3 months post implantation. The numberand size of the vessels within the remodeling UBM-ECM device werefurther decreased by 6 moths post implantation and resembled thevasculature found within the native TMJ meniscus. Both the number andthe size of the vessels observed were greater at the periphery of thedevice than in the bulk at all time points investigated.

B. CD68

Immunohistochemical staining for CD68 showed that a large number ofmononuclear macrophages were present within the dense cellularinfiltrate that was observed during the histologic evaluation of tissue3 weeks, 1 month, and 2 months post implantation. The number of CD68+macrophages decreased with time and few, if any, mononuclear cells wereobserved by 3 months post implantation. By 6 months post implantation,the dense mononuclear macrophage population observed at early timepoints was been replaced by a population of spindle shaped CD68− cellsresembling those found in the native TMJ meniscus. It should be notedthat the temporal and spatial patterns of the macrophage infiltrationseen in this study are the same those observed in numerous other studiesutilizing acellular ECM scaffold materials for tissue reconstruction.

Summary

These results show that the UBM-ECM device acted as an inductivetemplate for constructive remodeling of the TMJ meniscus followingmeniscectomy. The remodeling of the UBM-ECM device was characterized byinfiltration of a cell population consisting predominantly ofmononuclear macrophages accompanied by rapid degradation of the scaffoldmaterial, deposition of new host-derived tissue, and angiogenesis atearly time points changing to a sparse population of spindle shapedcells and small blood vessels within mature, highly aligned collagenwith time. By six months post implantation, the morphology of theremodeled ECM scaffold site highly resembled that of the native TMJmeniscus both in terms of its shape and size as well as its components,collagen fiber organization, and the makeup of the cell population. Theremodeled tissue was shown to be well-integrated with the nativemusculature at the periphery of the implant and host derived muscletissue was observed within the site of tissue remodeling. Further,implantation of the UBM-ECM device was not associated with anypathologic changes in the articulating surface of the fossa or condyleat any of the time points investigated.

Acellular, non-crosslinked ECM scaffolds are shown to induce theformation of new, site-appropriate, functional, host tissue that isarranged in a spatially appropriate pattern for the tissue of interest.This remodeling process is in direct contrast to the default mechanismof mammalian tissue repair following injury, which generally results ininflammation and scarring with no functional recovery. The exactmechanisms by which this ECM mediated tissue specific constructiveremodeling occurs are not fully understood; however, a number of factorsincluding mechanical forces, scaffold degradation with concomitantrelease of bioactive ECM molecules and matricryptic peptides, and theability of ECM scaffolds to modulate the host immune response are knownto play important roles in determining remodeling outcomes.

This is the first report of an intact ECM scaffold material used in anin vivo study of meniscus replacement. The non-crosslinked UBM-ECMdevice used in the present study was rapidly degraded and wasindistinguishable from newly deposited host tissue at the three weektime point, indicating that scaffold degradation in the TMJ locationoccurs very rapidly in this anatomic site. This rapid degradation may bedue, in part, to the highly complex synovial fluid milieu and themechanical loading environment of the TMJ location compared to otherlocations in which ECM scaffolds have been previously implanted. The TMJmeniscus has been shown to experience large tensile, compressive, andshear forces during everyday motion. These large and diverse forces mayhave contributed to the rapid degradation of the UBM-ECM scaffoldobserved in this study. Further, these site-specific forces may haveinfluenced the formation and resultant site appropriate spatialarrangement of fibrocartilage, vasculature, and muscle observed in thisstudy.

Example 4 Evaluation of TMJ Regeneration at Six (6) Months

Follow-up studies were conducted in light of the results of Example 3.Ten (10) dogs were used in this study. Each dog served as its owncontrol. All dogs had unilateral (left) and contralateral (right)meniscectomies of the TMJ meniscus. Contralateral TMJ menisci werereplaced with the UBM scaffold described in Example 3. After 6 monthsnecropsies were performed on the dogs. Menisci from four dogs have notyet been removed. The menisci were evaluated visually, and thenhistology, immunohistochemical staining, biochemical assays andbiomechanical testing were performed.

At the time of explant, the joint space of all animals was examined forsigns of pathologic degeneration of the articulating surfaces of thetemporal fossa and mandibular condyle and/or other signs of pathologicjoint inflammation. The UBM menisci also were examined. Grossmorphologic examination showed that there was little to no change in thearticulating surfaces of the temporal fossa or the mandibular condyle.There were no signs of synovitis, or excess fluid in the joint space.The UBM-ECM device was replaced with a structure that highly resembledthe fibrocartilage of the native TMJ disk. It was not possible todifferentiate the original UBM-ECM device from newly deposited hosttissue.

For histological studies, the explanted tissue were sectioned (see FIG.11) and slides were prepared and stained with Hematoxylin and Eosin asdescribed above. FIGS. 12A and 12B show normal Hematoxylin and Eosinstains for the UBM meniscus for dogs 1 and 2 respectively, and isexemplary of the results obtained for other subject dogs.

Von Kossa staining was performed on menisci of dogs 1 and 2. Briefly,paraffin sections are deparaffinized and hydrated with water; rinsedwith

distilled water; incubated with 1% silver nitrate solution and placedunder ultraviolet light for 20 minutes; incubated with 5% sodiumthiosulfate for 5 minutes to remove un-reacted silver; rinsed indistilled water; counterstained with nuclear fast red for 5 minutes;rinsed in distilled water; dehydrated in alcohol and xylene and acoverslip was applied. FIG. 13A shows gross morphology of the UBMmeniscus from Dog 1, which shows some apparent mineralization. Other UBMmenisci did not show evidence of mineralization to this extent, and mostshowed no evidence of mineralization. FIGS. 13B and 13C show the resultsof Von Kossa staining of sections of the UBM menisci from dogs 1 and 2,indicating that dog 1 had evidence of calcification, while dog 2 hadlittle or no mineralization. Because the mineralization is foundpredominantly in one sample, it is believed that the UBM implantationmethod affects the character of the UBM menisci. Where mineralization isfound it is believed bone progenitor cells contaminated the implant, andthat care needs to be taken not to contaminate the implant with bone orother non-cartilage progenitor cells during implantation.

Immunohistochemical analysis of markers of blood vessels and macrophages(CD31 and CD68, respectively) was performed essentially as described inExample 3. FIGS. 14A and 14B show CD31 staining for dogs 1 and 2,respectively. FIGS. 15A and 15B show CD68 staining for dogs 1 and 2,respectively.

Biomechanical and Biochemical Analysis

Four millimeter (4 mm) cylindrical punches were made for each meniscustested, leaving much of the tissue remaining for biochemical analysis.The cylindrical shape was chosen for simplicity of stress calculationsand modeling. The samples were placed in a temperature-controlled saline(0.9%) bath prior to testing. All testing was performed at 37° C. usingan MTS Insight testing system. Compression, relaxation and elasticmodulus were analyzed as shown below.

Unconfined compression analysis was performed by inputting a ramp strainuntil a given value (10% strain) was reached. Then compression was helduntil stress reaches equilibrium. FIGS. 16A and 16B are graphs showingthe compressive behavior for a native canine TMJ disc and for apre-implantation UBM construct, respectively. Of note, the UBM constructbehaves more elastically than the native disc. The native TMJ disc wasobserved to absorb water during unloading.

Percent relaxation over time was determined for native TMJ andpreimplantation UBM. In this method, a pre-load of 0.1N for 30 mins isapplied and the specimen height was determined. Then, preconditioningbetween 0-10% strain at 9%/min strain rate (slow) was performed. Thisyielded repeatable data. Then, stress-relaxation of 10% strain wasperformed and the sample was allowed to relax for 30 mins. FIGS. 17A and17B are graphs showing the relaxation data for native canine TMJ andpreimplanted UBM, respectively. Both showed very similar viscousbehavior, though the UBM implant had a higher elastic response. FIG. 18shows percent relaxation for native canine TMJ, pre-implantation UBM andremodeled UBM scaffold at 6 months. No significant differences inpercent relaxation were seen.

The Tangent Modulus is slope of linear region of stress-strain curve.Tangent Modulus was determined for native canine TMJ, pre-implantationUBM and remodeled UBM scaffold at 6 months. FIG. 19 is a graph showingthe Tangent Modulus. Notably, the remodeled UBM implant was twice asstiff as the native TMJ at 6 months post implantation; however theseresults were not statistically significant. These results suggest thatthe initially stiffer UBM implant is remodeling with mechanicalproperties that resembled those observed for the native meniscus at 6months post-implantation. The pre-implantation scaffold is much stifferthan native canine TMJ or remodeled UBM.

Biochemistry

The tested samples were analyzed for collagen, GAG and water content.Collagen content was assessed using a hydroxyproline assay, GAG contentwas assessed using a dimethylene blue assay, and water content wasassessed by measuring sample weight both pre- and post-lyophilization ofthe tissue to remove water content. FIG. 20 is a graph showing Collagenand GAG content for native canine TMJ, pre-implantation UBM andremodeled UBM scaffold at 6 months. FIG. 21 is a graph showing watercontent for native canine TMJ, pre-implantation UBM and remodeled UBMscaffold at 6 months. There was no significant difference in collagen,GAG and water content for all samples.

FIG. 22A is a tablulation of the data from Example 2. FIG. 22B providesaverages and standard deviation.

Example 5 Replacing a Human TMJ Meniscus with an ECM Scaffold

Patients whose TMJ meniscus is determined to be irreparably damaged, orthat failed previous meniscus replacement with other autogenous oralloplastic materials are considered for this procedure. The patient isplaced under a general anesthetic. The irreparably damaged human TMJmeniscus is surgically excised through a standard preauricular incision.Next, any adhesions on the undersurface of the glenoid fossa are removedwith a curette, and bony spurs on the articulating surface of thecondyle are conservatively removed with a rongeur. The articulatingeminence is smoothed of any irregularities via a bone file. Next, theappropriate size scaffold is surgically implanted with slow-resorbingsutures to both the root of the zygomatic arch and the adjacent muscletissue. The mandible is manipulated through a sterile drape to ensurethe appropriate fit and coordinated movement of the implant relative tothe condyle and fossa. The surgical field is irrigated with anantibiotic solution prior to being sutured in layers. The wound isclosed in layers and the patient is placed on an appropriate antibiotic(e.g., KEFLEX™, if not penicillin allergic) and analgesic (e.g., NSAID,and narcotic of the physician's choice for breakthrough pain) for sevendays. Physical therapy for the mandible is initiated by at least thefourth postoperative day.

Having described this invention, it will be understood to those ofordinary skill in the art that the same can be performed within a wideand equivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any embodiment thereof.

We claim:
 1. A bioscaffold for replacement or regeneration of atemporomandibular joint meniscus, the bioscaffold comprising: abiocompatible, biodegradable, elastomeric implant dimensioned to fitwithin the TMJ and mimic the size and function of a native TMJ meniscus,the implant comprising a top sheet formed from one or more layers of abiocompatible material, a bottom sheet formed from one or more layers ofthe biocompatible material, and a raised central core consistingessentially of an acellular and noncrosslinked extracellular matrixdisposed between the top and bottom sheets, wherein the top sheet andbottom sheet have substantially the same thickness, and further whereinthe top and bottom sheets radially extend from the raised central coreto form a brim having surgical attachment sites in the brim; wherein thecentral core has a circular base with a diameter, or an elliptical basewith a major diameter and a minor diameter, and a height and compositionof the central core that permits the core to deform under stress, andthe diameter or the major and minor diameter is sufficient for the coreto carry shear forces between the mandible and the skull when theimplant is affixed to an articulating surface in the TMJ.
 2. Thebioscaffold of claim 1, wherein at least one of the top or bottom sheetsis formed from two layers of biocompatible material encapsulating thecore.
 3. The bioscaffold of claim 1, wherein the core is encased in aplurality of layers of extracellular matrix.
 4. The bioscaffold of claim1, wherein the core comprises a gel or particulate extracellular matrixthat is encased in a plurality of layers of extracellular matrix,wherein the gel or particulate extracellular matrix within the coreallows compression of the core.
 5. The bioscaffold of claim 4, whereinthe core comprises the particulate extracellular matrix encased in theplurality of layers of extracellular matrix.
 6. The bioscaffold of claim5, wherein the particulate extracellular matrix of the core comprisesparticles having a particle size from 10 to 400 microns.
 7. Thebioscaffold of claim 6, wherein the particles have an average particlesize of 150 to 200 microns.
 8. The bioscaffold of claim 1, wherein thebioscaffold additionally comprises a synthetic polymer that impartsflexibility to the bioscaffold.
 9. The bioscaffold of claim 1, whereinthe core is concave, convex, biconcave, biconvex, or a combinationthereof.
 10. The bioscaffold of claim 1, wherein both the core and thetop and bottom sheets comprise acellular and noncrosslinkedextracellular matrix of tissue selected from the group consisting ofsmall intestine, urinary bladder, esophagus, skin, liver, spleen, heart,pancreas, ovary, and arteries.
 11. The bioscaffold of claim 1, whereinthe central core has an elliptical base with a major diameter and aminor diameter.
 12. A bioscaffold for replacement or regeneration of atemporomandibular joint meniscus, the bioscaffold comprising: abiocompatible, biodegradable, elastomeric implant dimensioned to fitwithin the TMJ and mimic the size and function of a native TMJ meniscus,the implant comprising a top sheet formed from one or more layers ofbiocompatible material, a bottom sheet formed from one or more layers ofbiocompatible material, and a central core disposed between the top andbottom, wherein the core comprises acellular and noncrosslinkedextracellular matrix particles or gel and the core extends between thetop or bottom sheets to form a nonplanar pillow having a height ofbetween 0.5 and 4 cm, wherein the core has a circular base with adiameter, or an elliptical base with a major diameter and a minordiameter, and the height and composition of the core permits the core todeform under stress, and the diameter or major and minor diameter of thecore is sufficient for the core to carry shear forces between themandible and the skull when the implant is affixed to an articulatingsurface in the TMJ; and wherein the top sheet and bottom sheet extendoutwardly from the core to form a brim with surgical attachment sites inthe brim.
 13. The bioscaffold of claim 12, wherein the core comprises aplurality of layers of extracellular matrix.
 14. The bioscaffold ofclaim 13, wherein the core pillow is shaped concave, convex, biconcave,biconvex, or a combination thereof.
 15. The bioscaffold of claim 14,wherein the core comprises a convex pillow extending from the base. 16.The bioscaffold of claim 15, wherein the core comprises a deformablegel.
 17. The bioscaffold of claim 16, wherein the extracellular matrixis an extracellular matrix gel that gels at a temperature at or above 25degrees Celsius.
 18. The bioscaffold of claim 12, wherein core comprisesdeformable particulate extracellular matrix particles having a particlesize from 10 to 400 microns.
 19. The bioscaffold of claim 18, whereinthe particles have an average particle size of 150 to 200 microns. 20.The bioscaffold of claim 12, additionally comprising a syntheticelastomeric polymer.
 21. The bioscaffold of claim 12, wherein both thecore and the top or bottom sheets comprise extracellular matrix oftissue selected from the group consisting of small intestine, urinarybladder, esophagus, skin, liver, spleen, heart, pancreas, ovary, andarteries.
 22. The bioscaffold of claim 12, wherein at least a portion ofthe top or bottom sheets comprises basement membrane.
 23. Thebioscaffold of claim 12, wherein the at least one of the top or bottomsheets has an inner surface and an outer surface, and wherein at least aportion of the outer surface comprises basement membrane.
 24. Abioscaffold for replacement or regeneration of a temporomandibular joint(TMJ) meniscus the bioscaffold comprising: a biocompatible,biodegradable, elastomeric implant dimensioned to fit within the TMJ andmimic the size and function of a native TMJ meniscus, the implantcomprising a top sheet formed from one or more layers of biocompatiblematerial, a bottom sheet formed from one or more layers, and a raisedcentral elastomeric core comprising acellular and noncrosslinkedextracellular matrix particles or gel disposed between the top andbottom sheets, wherein the core is deformable under stress to cushionforces between the mandibular condyle and the skull bones with which itarticulates when the implant is affixed to an articulating surface inthe TMJ and wherein the top and bottom sheets radially extend from thecore to form a brim comprising one or more surgical attachment sites;wherein the core is 8-12 mm wide, and the implant is 16-20 mm wide; andwherein the core comprises a circular base with a diameter or anelliptical base with a major diameter and a minor diameter, whereby thecore can carry shear forces between the mandible and skull when theimplant is affixed to the articulating surface.
 25. The bioscaffold ofclaim 24, wherein the core comprises particulate extracellular matrixand is encased in a plurality of layers of extracellular matrix.
 26. Thebioscaffold of claim 24, wherein the core extends from the base in ashape that is concave, convex, biconcave, biconvex, or any combinationthereof.
 27. The bioscaffold of claim 24, wherein core comprisesparticulate extracellular matrix having a particle size from 10 to 400microns.
 28. The bioscaffold of claim 27, wherein the particulateextracellular matrix has an average particle size of 150 to 200 microns.29. The bioscaffold of claim 27, additionally comprising a syntheticelastomeric polymer.
 30. The bioscaffold of claim 24, wherein theextracellular matrix is an extracellular matrix gel that gels at atemperature at or above 25 degrees Celsius.
 31. A bioscaffold forreplacement or regeneration of a temporomandibular joint meniscus, thebioscaffold comprising: a biocompatible, biodegradable, elastomericimplant dimensioned to fit within the TMJ and mimic the size andfunction of a native TMJ meniscus, the implant comprising a top sheetformed from one or more layers of a biocompatible material, a bottomsheet formed from one or more layers of the biocompatible material, anda central core comprising an acellular and noncrosslinked extracellularmatrix gel, or particles having a particle size from 10 to 400 microns,disposed between the top and bottom sheets, wherein the top sheet andbottom sheet have substantially the same thickness, and further whereinthe top and bottom sheets extend around the central core to form a brimhaving surgical attachment sites in the brim; wherein the central corehas an elliptical base with a major diameter and a minor diameter, andthe central core extends away from the brim in a shape that is concave,convex, biconcave, biconvex, or any combination thereof and a height andcomposition of the central core permits the core to deform under stress,and central core with the elliptical base is elongated along the majordiameter for the core to carry shear forces between the mandible and theskull when the implant is affixed to an articulating surface in the TMJ.32. The bioscaffold of claim 31 wherein the central core comprises theparticles, wherein the particles have an average particle size of 150 to200 microns.
 33. The bioscaffold of claim 32, wherein the central coreextends 0.4 to 4 cm from the brim, has a major diameter of 8-12 mm, andthe implant is 16-20 mm wide.